Viable but nonculturable (VBNC) cells were recognized 30 years ago; and despite decades of research on the topic, most results are disperse and apparently incongruous. Since its description, a huge controversy arose regarding the ecological significance of this state: is it a degradation process without real significance for bacterial life cycles or is it an adaptive strategy of bacteria to cope with stressful conditions? In order to solve the molecular mechanisms of VBNC state induction and resuscitation, researchers in the field must be aware and overcome common issues delaying research progress. In this review, we discuss the intrinsic characteristic features of VBNC cells, the first clues on what is behind the VBNC state's induction, the models proposed for their resuscitation and the current methods to prove not only that cells are in VBNC state but also that they are able to resuscitate.

The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37°C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15°C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.

Aims:  To analyse viable but nonculturable (VBNC) state induction in Escherichia coli and resuscitation of VBNC suspensions in several conditions. Methods and Results:  VBNC were induced in four media, two temperatures and six strains, but only cells produced at 4°C were able to resuscitate. Resuscitation of 14 VBNC suspensions obtained in several conditions occurred in the presence of supernatants of growing cells, in minimal medium supplemented with amino acids or after temperature change, depending on strain. A limited time period beyond no more resuscitation that could be observed was also confirmed. Conclusions:  The supernatants positive effect is suggested to be because of a nonproteinaceous molecule, and a combination of methionine, glutamine, threonine, serine and asparagine could be used as primary mix for resuscitation experiments. Significance and Impact of the Study:  Escherichia coli resuscitation was already attempted in several conditions, but it is the first time that a positive result was observed in minimal medium supplemented with amino acids or after temperature change. The role of amino acids in resuscitation is of special interest since was never reported for any species.

The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.All tailed bacteriophages with double-stranded DNA genomes appear to accomplish lysis of the host cell by the concerted action of a peptidoglycan hydrolase (referred to as endolysin or lysin) and a small hydrophobic protein (holin) presumed to form nonspecific lesions upon oligomerization in the membrane (for a review, see reference 41). The latter function seems essential to allow access of the lytic enzyme to the cell wall compartment since in the phage lysins examined so far, the presence of a signal peptide (SP) that would target them to the translocase of the general secretion pathway (GSP) has never been demonstrated.We have recently described the sequences of the lysin and holin genes from the Oenococcus oeni bacteriophage fOg44 and noted that the N-terminal region of its putative lysin (Lys44) was highly hydrophobic (23). A similar observation was made earlier concerning a related enzyme from the lactococcal phage Tuc2009 (2). In spite of its hydrophobic character, the function of the N-terminal sequence of the Tuc2009 lysin as a possible SP was not considered, presumably because the presence of a holin gene upstream of lys argued for a standard holin-dependent lysin export mechanism. This assumption was strengthened by the observation that the expression of an almost identical lysin in Escherichia coli (LysB from the Lactococcus lactis phage LC3) did not result in a decrease in culture absorbance unless the corresponding holin was simultaneously induced (3).Interestingly, however, during an attempt to overproduce Lys44 in an easily purifiable form (as a histidine-tagged fusi...

The results reported here have identified yueB as the essential gene involved in irreversible binding of bacteriophage SPP1 to Bacillus subtilis. First, a deletion in an SPP1-resistant (pha-2) strain, covering most of the yueB gene, could be complemented by a xylose-inducible copy of yueB inserted at amyE. Second, disruption of yueB by insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-␤-D-thiogalactopyranoside). YueB homologues are widely distributed in grampositive bacteria. The protein Pip, which also serves as a phage receptor in Lactococcus lactis, belongs to the same family. yueB encodes a membrane protein of ϳ120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain. Insertional inactivation of yueB and the surrounding genes indicated that yueB is part of an operon which includes at least the upstream genes yukE, yukD, yukC, and yukBA. Disruption of each of the genes in the operon allowed efficient irreversible adsorption, provided that yueB expression was retained. Under these conditions, however, smaller plaques were produced, a phenotype which was particularly noticeable in yukE mutant strains. Interestingly, such reduction in plaque size was not correlated with a decreased adsorption rate. Overall, these results provide the first demonstration of a membrane-bound protein acting as a phage receptor in B. subtilis and suggest an additional involvement of the yukE operon in a step subsequent to irreversible adsorption.The interaction of a bacteriophage with the bacterial surface (adsorption) is the first step in the infection process. This step involves recognition of and binding to one or more cell envelope constituents and leads to ejection of the phage DNA from the capsid. It has long been appreciated that adsorption can proceed in two steps, one reversible step followed by irreversible commitment (1). Irreversible adsorption and ejection of DNA are possibly different descriptions of the same phenomenon, but this has not really been clarified. Similarly, DNA ejection from the capsid and injection into the cytoplasm are at best difficult to separate in vivo, except in those cases where injection itself proceeds in distinct phases, as in T5 (13).In the gram-negative world, several phage receptors have been identified, and in a number of cases (e.g., T4, T5, and T7 of Escherichia coli), the adsorption and injection processes are now beginning to be understood at a detailed molecular level (14,18,26). Much less is known about the first steps of infection for phages preying on gram-positive bacteria. Early work has established that glucosylated polyglycerol phosphate, the major and essential teichoic acid in Bacillus subtilis, serves as a receptor for several phages, including 29, 25, and SP01 (24,29,34,36). Mutations leading to a lack of glucosylation of this polymer block the phage adsorption process and plaque-forming ability. More rece...